The invention relates to compounds containing boron, their production and the use thereof for the energy-filtering transmission electron microscopy and for the boron neutron-capture therapy.
It is known to detect boron-containing compounds by the energy-filtering transmission electron microscopy (EFTEM).
For example, Qualmann B. et al., Angew. Chem., 1996 180, pages 970 to 973, describes the synthesis of boron-containing lysine dendrimers for protein labeling in electron microscopy. The boron-containing compound described in this publication and serving for labeling proteins is unsuitable for labeling small biological molecules, e.g. oligonucleotides, because of its large expansion, since the material properties of such small biological molecules are modified excessively. Furthermore, because of the large expansion of the compound and the arrangement of the 1,2-dicarbadodecaborane fragments (carboranes) in the outermost sphere of the molecule the boron density is very low. Therefore, a specification of this compound by means of EFTEM is not possible to a satisfactory extent. In addition, the described compound has a peptide base structure with L-lysine as building blocks, so that it is susceptible to enzymatic degradation.
The publication by Newkome G. R. et al., in Angew. Chem., 1994, 106, pages 701 to 703, describes unimolecular micelles, which contain 4 or 12 carboranes in the micelle interior and hydrophilic groups on the surface of the micelles. These unimolecular micelles are also too big. Furthermore, no binding site exists for the linkage of a spacer for attachment to biomolecules, such as oligonucleotides and proteins.
Therefore, it was formerly not possible to label for detection by EFTEM small biologically active substances, such as oligonucleotides, by a boron-containing compound to be bonded covalently.
It is also known to use boron-containing compounds in the boron neutron-capture therapy. However, it was not yet possible to selectively introduce sufficiently high boron concentrations into the tumor tissue.
Thus, the object of this invention is to provide a compound which does not show the drawbacks of the prior art.
According to the invention this is achieved by the subject matters defined in the claims.
The subject matter of the present invention relates to a boron-containing compound which has the following general formula (1) 
in which
Cb stands for a carborane,
R2 and R3 are independently a hydrogen atom or an organic residue, and
R and R1 are independently a hydrogen atom or an organic residue with the carbon atom to which they are bound.
The expression xe2x80x9ccarboranexe2x80x9d comprises compounds of any kind, which include the summation formula B10C2H12. The carboranes may form three isomers: 1,2-dicarba-closo-dodecaborane, 1,7-dicarba-closo-dodecaborane, and 1,12-dicarba-closo-dodecaborane, which are also referred to as ortho-, meta-, and para-carborane. Of these isomers the 1,2-dicarba-closo-dodecaborane is preferred, which is symbolized as follows: 
Because of the two carbon atoms the carboranes have two possible binding sites with other compounds. Due to the linkage of the four carboranes with the hydrocarbon skeleton indicated in formula (1) one binding site of the carborane is occupied. The second binding site can now be used for binding spacers and/or solubility-modulating compounds.
The expression xe2x80x9cspacerxe2x80x9d comprises compounds of any kind, which can be used for linkage, in particular for covalent linkage, of the boron-containing compound according to the invention with other molecules, e.g. with biological molecules. Such compounds are known to a person skilled in the art. They are preferably compounds derived from C2 to C10 alkanes, in particular C6 alkanes, which are preferably linear and optionally have ether bridges or may be bound via such a bridge to the carborane. The expression xe2x80x9cbiological moleculesxe2x80x9d refers to the fact that any kind of molecule relevant for biological processes is concerned. Examples are proteins, nucleotides, such as mono-, oligo-, and polynucleotides, nucleosides, nucleoside diphosphates and nucleoside triphosphates. Of the proteins those are preferred which accumulate in tumors, such as albumin.
The xe2x80x9csolubility-modulating compoundsxe2x80x9d are compounds of any kind, which raise or lower the solubility of the compound according to the invention in a solvent, in particular water or an aqueous solvent. To raise the water solubility, the solubility-modulating compounds may have at least one polar group, such as a hydroxyl group. Examples thereof are xe2x80x94CH2OH and polyhydroxy compounds, such as inositol or saccharides, in particular monosaccharides, preferably glucose. Because of the high degree of lipophilia of the carboranes and the hydrocarbon skeleton the bonding of solubility-modulating compounds is favorable for raising the water solubility of the boron-containing compound.
By the use of carbohydrates, in particular glucose, galactose, xylose, fucose or also gentiobiose the tumor selectivity of the boron-containing compound according to the invention can also be increased.
Examples of compounds in which glucose is bound to the carboranes are the compounds shown in formulae (2) and (3). 
This serves for obtaining small lipophilic molecule cores in which the entire boron amount is concentrated and a hydrophilic molecule shell which increases the water solubility of the boron-containing compound and optionally the tumor selectivity. All in all, a unimolecular micelle is thus obtained.
In the boron-containing compound according to the invention, the substituents R2 and R3 are independently a hydrogen atom or an organic residue. The expression xe2x80x9corganic residuexe2x80x9d covers organic compounds of any kind, which comprise carbon, hydrogen and optionally oxygen, sulfur, phosphorus and boron. Examples thereof are the groups xe2x80x94NO2, xe2x80x94(Cxe2x95x90O)xe2x80x94, xe2x80x94Cxe2x89xa1Nxe2x80x94, phenyl and xe2x80x94COOR4, wherein R4 represents e.g. an alkyl residue such as ethyl. R2 and R3 can also be linked with each other, i.e. the residues denoting R2 and R3 are chosen such that they form a ring, preferably a 6-membered ring, with the carbon atoms to which they are directly bound and with the carbon atom including the residues R and R1. Further carboranes can be bound to the ring, so that the total number of carboranes in the compound according to the invention is increased, e.g. to 6. Examples of R2 and R3 for forming a 6-membered ring are: 
In the boron-containing compound according to the invention, the substituents R and R1 are independently a hydrogen atom, an organic group or R and R1 form a carbonyl group together with the carbon atom to which they are bound. The organic group can be every compound containing carbon, hydrogen and optionally oxygen, sulfur, phosphorus and boron. For example, the group is a C2 to C10, in particular C6, alkyl group bound via an ether bridge. A phosphate group (PO43xe2x88x92) can be bound to the alkyl group, in particular to the end thereof. The phosphate group can be bound to a biological molecule, such as a poly-, oligo- or mononucleotide, preferably at the 51 ends thereof. An example of such an organic group standing for R and R1, respectively, is shown in above formula (3).
Another subject matter of the present invention relates to a method of producing the boron-containing compounds according to the invention, in which a decaborane is reacted with an alkyne of formula (4). 
A decaborane is a compound with the summation formula B10H14, which differs from the above described carboranes in that inter alia the two carbon atoms are not present. They are introduced by the two carbon atoms of the alkyne bond.
It was found surprisingly that this reaction will also be possible if at least four carboranes are bound to a very small hydrocarbon base structure in spite of the steric impediment. It was also found that surprisingly high yields will be obtained if a carbonyl group is disposed in the xcex2-position relative to the alkyne group. If necessary, the carbonyl group can be protected as usual and then be deprotected also in known manner.
Examples of this reaction are shown in the reaction equations (I) to (VI). 
The alkyne compounds can be produced from CH-acidic carbonyl compounds in a manner with which the person skilled in the art is familiar. This is carried out e.g. by means of C alkylation of enolates of esters or ketones which can be produced by deprotonation using suitable bases, such as aqueous caustic soda solution, under phase transfer catalysis or sodium methylate in methanol and are reacted with a proparyl halide, such as proparyl bromide.
The resulting boron-containing compounds according to the invention offer per carborane a further position for introducing the above-mentioned spacers or solubility-modulating compounds, such as saccharides, in particular glucose. The carbonyl group(s) can then be used for binding an above described spacer and thus for linkage to biological molecules and solubility-modulating compounds. For this purpose, the carbonyl group can be reduced with respect to the alcohol, e.g. by means of LiAlH4 followed by the formation of an ether group.
Alkynes which already have side chains for the linkage of biological molecules, e.g. saccharides, can also be used for the process according to the invention for producing boron-containing compounds. Such a starting compound is indicated in formula (5). 
In this connection, the biological molecules can be attached to the hydroxyl group(s). For this reaction it proved to be favorable to protect the four OH groups prior to the reaction with the decaborane (14) and deprotect them after the reaction. Every protective group known for OH groups, e.g. an ester group, such as an acetate group, can be used as a protective group. The deprotection is made in known manner, depending on the protective group used.
The compounds according to the invention have a number of advantages: The spatial expansion of the compounds according to the invention is very little, so that the properties of the biological molecules, e.g. the oligonucleotides, are not disturbed. Furthermore, a high degree of stability of the boron-containing compound is achieved by the linkage of the carboranes by the narrow (small) hydrocarbon skeleton. In addition, the compound according to the invention can be defined accurately and can clearly be positioned on a biological molecule. Furthermore, the properties of the boron-containing compounds can be modified widely by the spacer and the solubility-modifying compound. This leads to a diverse usability for the covalent bond to biological molecules, such as proteins, monomeric nucleoside triphosphates, and oligo- and polynucleotides, so that the boron-containing compounds according to the invention are perfectly suited for the energy-filtering transmission electron microscopy. The boron-containing compounds according to the invention can also perfectly be used for the boron neutron-capture therapy to treat tumors, since sufficient boron density is achieved, on the one hand, and by linkage with saccharides it is also possible to produce excellent tumor selectivity by either uptake via glucose transporters (GLUTs or SGLTs) or lectin-mediated endocytosis, on the other.